Increasing The Production Of Recombinant Antibodies In Mammalian Cells By Site-Directed Mutagenesis

ABSTRACT

The present invention relates to a reliable, reproducible method for improving the producibility of an antibody. More specifically, this invention provides a method for modifying the heavy chain of an antibody to improve its producibility in eukaryotic cells. Additionally, the method of the invention may improve both antibody producibility and one or more antigen binding characteristics. The invention further provides modified antibodies which are better produced and which have either no change in their antigen binding characteristics or exhibit improved antigen binding characteristics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/165,023, filedJun. 24, 2005, said application Ser. No. 11/165,023 claims the benefitunder 35 U.S.C. §119(e) of the following U.S. Provisional ApplicationNos. 60/583,184, filed Jun. 25, 2004 and 60/624,153, filed Nov. 2, 2004.The priority applications are hereby incorporated by reference herein intheir entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled “AE700US_ST25.TXT” createdJun. 24, 2005 and having a size of 32 kilobytes.

BACKGROUND OF THE INVENTION

The use of antibodies to block the activity of foreign and/or endogenouspolypeptides provides an effective and selective strategy for treatingthe underlying cause of disease. In particular is the use of recombinantmonoclonal antibodies (MAb) and antibody fragments as effectivetherapeutics such as the FDA approved Synagis (Saez-Llorens, X. E., etal., 1998, Pediat. Infect. Dis. J. 17:787-91), an anti-respiratorysyncytial virus MAb produced by Medimmune; ReoPro (Glaser, V., 1996,Nat. Biotechnol. 14:1216-17), an anti-platelet Fab antibody fragmentfrom Centocor; and Herceptin (Weiner, L. M., 1999, Semin. Oncol.26:43-51), an anti-Her2/neu MAb from Genentech.

Standard methods for generating MAbs against candidate protein targetsare known by those skilled in the art. Briefly, rodents such as mice orrats are injected with a purified antigen in the presence of adjuvant togenerate an immune response (Shield, C. F., et al., 1996, Am. J KidneyDis. 27:855-64). Rodents with positive immune sera are sacrificed andsplenocytes are isolated. Isolated splenocytes are fused to melanomas toproduce immortalized cell lines that are then screened for antibodyproduction. Positive lines are isolated and characterized for antibodyproduction. However, the use of rodent MAbs directly as humantherapeutic agents may result in the production of the human anti-rodentantibody (HAMA) response (Khazaeli, M. B., et al., 1994, J Immunother.15:42-52). This response reduces the effectiveness of the antibody byneutralizing the binding activity and by rapidly clearing the antibodyfrom circulation in the body. The HAMA response can also causesignificant toxicities with subsequent administrations of rodentantibodies.

In order to reduce the HAMA response the production of human-murinechimeric antibodies in which the genes encoding the mouse heavy andlight chain variable regions have been coupled to the genes for humanheavy and light chain constant regions to produce chimeric or hybridantibodies is commonly utilized. In some cases, mouse CDRs have beengrafted onto human constant and framework regions with some of the mouseframework amino acids being substituted for correspondingly positionedhuman amino acids to provide a “humanized” antibody. Examples detailingthe production of chimeric and/or humanized antibodies can be found inJordan et al. U.S. Pat. No. 6,652,863; Winter et al. U.S. Pat. No.5,225,539; Queen et al. U.S. Pat. Nos. 5,693,761 and 5,693,762; andAdair et al. U.S. Pat. No. 5,859,205, which are incorporated herein byreference in their entirety

Human antibodies can also be generated and “matured” by screening phagedisplay antibody libraries derived from human immunoglobulin sequences.Techniques and protocols required to generate, propagate, screen (pan),and use the antibody fragments from such libraries have recently beencompiled (See, e.g., Barbas et al., 2001, Phage Display: A LaboratoryManual, Cold Spring Harbor Laboratory Press and Kay et al. (eds.), 1996,Phage Display of Peptides and Proteins: A Laboratory Manual, AcademicPress, Inc., also see, Winter et al. U.S. Pat. No. 6,225,447 and Knappiket al. U.S. Pat. No. 6,300,064; Kufer et al. PCT publication WO98/46645; Barbas et al. U.S. Pat. No. 6,096,551; and Kang et al. U.S.Pat. No. 6,468,738 each of which is incorporated herein by reference inits entirety.) Typically, phage-displayed antibody fragments are scFvfragments or Fab fragments; when desired, full length antibodies can beproduced by cloning the variable regions from the displaying phage intoa complete antibody and expressing the full length antibody in aprokaryotic or a eukaryotic host cell.

As glycoproteins, antibodies typically include oligosaccharide(carbohydrate) chains attached to the protein at specific amino acidresidues. The number, type, and location of the carbohydrate attachmentson the protein can affect key properties of commercialbiopharmaceuticals including clearance rate, immunogenicity, biologicalspecific activity, solubility and stability against proteolysis. Humanswill typically accept only those biotherapeutics that have particulartypes of carbohydrate attachments and will often reject glycoproteinsthat include non-mammalian oligosaccharide attachments. As a result,eukaryotic cells such as yeast and mammalian cell lines (e.g., ChineseHamster Ovary (CHO), Baby Hamster Kidney (BHK), Human EmbryonicKidney-293 (HEK-293)) are used for the production of the vast majorityof these glycoprotein therapeutics because of their capacity to generateglycoforms and perform other post-translational processing patterns thatare accepted by human patients. Unfortunately, production ofbiotherapeutics in mammalian cells can be expensive due to the need togrow these cells in costly cell culture environments and becausemammalian cells often produce the proteins in low yields. Thus,expression and production of the engineered antibody is the next hurdlethat must be over come for manufacturing of the molecule for clinicalmaterials.

Methods for producing a larger amount of monoclonal antibodies bymanipulating the culture conditions have been reported. For example,McCormack et al. (1988, Cell immunol. 115:325-33) reported that antibodyproduction increases when human antibody-producing hybridomas arecultured in an interleukin 2-supplemented culture medium, Grunberg etal. (2003, Biotechniques 34:968-72) demonstrate that the addition ofsodium butyrate can increase the expression of recombinant antibodyfragments from HEK-293 cells while Knibbs et al. (2003, Biotechnol Prog.19:9-13) describe the use of hillex microcarrier beads to increase theyield of antibodies from COS-7 cells. However, culture medium basedmethods such as these do not address the issue of antibody stability andsolubility, crucial factors influencing antibody expression andproduction yields.

Antibody folding efficiency and stability of the antibody fragmentsoften severely limit actual production levels. Thus, it is desirable toincrease expression yields by directly engineering the antibody moleculeto improve these characteristics. However, the factors influencingantibody stability and expression are still only poorly understood.

Some progress has been made in bacterial systems. For example, Ulrich etal. (1995, Proc. Natl. Acad. Sci. USA 92:11907-11) found that pointmutations in the complementarily determining regions (CDRs) can increasethe yields of Fab fragments in bacteria. Similarly, Plückthun andcolleagues (Knappik et al., 1995, Protein Engng. 8:81-9; Wall et al.,1999, Protein Engng. 12:605-11; Ewert et al., 2003, Biochemistry42:1517-28 and European. Pat. No. 0938506) showed that primary aminoacid sequence can influence folding efficiency and thus production ofimmunoglobulin (Ig) fragments in E. coli. In addition, Plückthun et al.(European. Pat. No. 0938506 and Ewert et al., 2003, Biochemistry42:1517-28) disclose a method to improve the solubility and the yield ofIg domains in bacterial systems by making the domain interface morehydrophilic. However, this method is very time consuming. Furthermore,the procedure requires a detailed knowledge and understanding of the3-dimensional structure of Ig domains and involves the use of expensivecomputer modeling programs to predict changes that may lead to astabilized Ig domain.

All of the studies described supra are limited to the expression of Igfragments and one would not predict that similar point mutations wouldhave a similar effect on folding, stability or expression of an intactantibody. In addition, many of the mutations described fall within theCDRs and could be expected to reduce the affinity or even alter thespecificity of an antibody. Furthermore, the studies described supra arelimited to expression of immunoglobulin fragments in bacterial systems,specifically E. coli. Human cells and other eukaryotes are subdivided bymembranes into many functionally distinct compartments, unlikebacterium, which exist as a single compartment surrounded by a membrane.Eukaryotic cells use “sorting signals,” which are amino acid motifslocated within the protein, to target proteins to particular cellularorganelles. One type of sorting signal, called a signal sequence,directs proteins destined for the membrane and/or secretion to anorganelle called the endoplasmic reticulum (ER). Antibodies fold andassemble after they are directed into the ER aided by a special class ofproteins called chaperones (e.g., Hsp70 (BiP), Hsp90 (GRP94) (Melnick etal., 1994, Nature 370:373-5) and Erp72 (Wiest et al., 1990, J. CellBiol. 110:1501-11). In addition, protein disulfide isomerase (PDI) isinvolved in the generation of the stabilizing disulphide bonds. Incontrast, the chaperone content of the periplasmic space of bacterium isfar more modest and there is no evidence for ATP in this compartment. Infact, Plüchthun indicates that while primary protein structure is themost important factor in E. coli it is the chaperone proteins that areimportant factors affecting folding, and thus production, in eukaryotes,(see the discussion section of Knappik et al., 1995, Protein Engng.8:81-9). Thus, one would not expect that protein alterations increasingIg domain production in bacterial systems (e.g., E. coli) would beapplicable to the expression and production of full length antibodies ineukaryotic cells.

Park et al. (U.S. Pat. No. 6,455,677) disclose certain frameworkmodifications of a FAPa-specific antibody, which improve theproducibility of this antibody. However, the methodology used was timeconsuming requiring the screening of numerous mutations as well as lightand heavy chain combinations. Additionally, they did not demonstratedthat the modifications made would be widely applicable to otherantibodies.

Steipe et al. (U.S. Pat. No. 6,262,238) disclose a different approachfor antibody stabilization involving amino acid substitutions in thevariable domain of the light and/or heavy chains. However, this approachrequires the substitution of numerous amino acids without a clearindication of which are important for stabilization of the antibody.Furthermore, alterations of the variable domains of antibodies can havedeleterious effects on the binding specificity and/or affinity of thealtered antibody. Mutations that alter the binding specificity or reducethe affinity of an antibody may render it clinically and thereforecommercially worthless. Thus, this approach involves laborious screeningto identify those mutations, which stabilize the antibody withoutnegatively affecting the binding affinity or specificity.

In many instances recombinant expression of native, chimeric and/orCDR-grafted antibodies in mammalian cell culture systems is poor due toimproper folding and reduced secretion. Improper folding can lead topoor assembly of heavy and light chains or a transport incompetentconformation that forbids secretion of one or both chains. It isgenerally accepted that the light chain confers the ability of secretionof the assembled protein in eukaryotic cells, as it is required for therelease of the heavy chain from BiP (Lee et al., 1999, Mol Biol Cell.,10:2209-19; Vanhove et al., 2001, Immunity 15:105-14). Given theimportant role of the light chain in assembly and secretion ofantibodies one would not have predicted that substitutions in the heavychain alone would dramatically increase antibody production in mammaliancells.

While the market for monoclonal antibodies is estimated to grow 30% ayear and reach sales of nearly $24 billion by 2010 there is a severeshortage of antibody manufacturing capacity (Garber, 2001, Nat Biotech.19:184-5). Another serious limitation relating to the commercial use ofantibodies is their producibility in large amounts. Many antibodies withtherapeutic or commercial potential are not expressed efficiently andcannot be developed due to inherent production limits. The producibilityof an antibody is determined by a large number of factors including butnot limited to, the host cell used, the growth conditions, the level ofgene expression, the stability of the messenger RNA, the stability ofthe translated antibody protein, protein folding, level of proteinaggregation, and the toxicity of the antibody to the host cell. Whileprogress has been made in understanding how some of these factorsinfluence the overall producibility of an antibody, few methods havebeen developed that lead to a reliable or reproducible increase inproducibility of any antibody. Thus, there is a real need for a rapidand reproducible method for increasing the producibility of recombinantantibodies for both clinical development and pharmaceuticalmanufacturing.

The present invention provides for the first time an antibodyengineering method that will reproducibly increase antibody productionin eukaryotic cells (e.g., mammalian cell lines) without resulting in asignificant negative effect on the binding characteristics of themodified antibody. The method of the present invention eliminates theneed for costly and time consuming random mutagenesis techniques thatcan result in an antibody with altered binding affinity and/orspecificity while reliably increasing antibody production fromeukaryotic cells.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

The inventors have made the surprising discovery that specific residuesof the immunoglobulin heavy chain play an important role in theproducibility (e.g., production levels, yield; expression levels) ofantibodies in eukaryotic systems. The inventors have further determinedthat the substitution of these amino acid residues results in anantibody that is produced at significantly higher levels than theunmodified antibody. Although not intending to be bound by any mechanismof action, the amino acid residue substitutions of the invention mayresult in an increase in antibody productivity by altering any or all ofa number of factors known to affect antibody producibility including butnot limited to, the level of gene expression, mRNA turnover and/ortranslation, antibody stability, antibody folding, antibody secretion,antibody aggregation, and the toxicity of the antibody to the host cell.

Mutations of the CDRs can have an adverse affect on the antigen bindingproperties of an antibody, however, the inventors have foundunexpectedly, that certain substitutions in the CDRs that enhanceproducibility did not negatively affect antigen binding and couldactually enhance the antigen binding properties of the modifiedantibody. Without wishing to be bound by any particular theory, theamino acid residue substitutions of the invention may result inconformational changes that include, but are not limited to, those thathave little or no effect on the antigen binding, those that result in anacceptable decrease in antigen binding, and those that result in animprovement in antigen binding.

Accordingly, the present invention provides a novel method forincreasing the producibility of antibodies or antibody fragments andprovides novel antibody sequences of the same. Also provided by thepresent invention are antibodies having at least one amino acid residuesubstitution, wherein the producibility of said substituted antibody isimproved compared to the antibody without said substitution.

The method of the invention involves changes of at least one residue ofthe heavy chain of an antibody of interest which lead to a significantincrease in production and which also may improve the antigen bindingcharacteristics of the antibody.

The present invention provides a method for increasing the producibilityof an antibody or antibody fragment comprising the steps of: (a)substituting the amino acid residues at positions 40H, 60H, and 61H,utilizing the numbering system set forth in Kabat, of the antibody ofinterest with alanine, alanine and aspartic acid, respectively; and (b)cultivating the host cell under conditions where the modified antibodypolypeptide is expressed by said host cell.

It is specifically contemplated that one skilled in the art may chooseto analyze the nature of the amino acids at positions 40H, 60H and 61Hof the antibody of interest prior to making any substitutions at thesepositions.

In a preferred embodiment, the host cell is eukaryotic includingeukaryotic microbes such as yeast. In a more preferred embodiment thehost cell is mammalian. Such mammalian host cells include but are notlimited to, CHO, BHK, HeLa, COS, MDCK, NIH 3T3, W138, NSO, SP2/0 andother lymphocytic cells, and human cells such as PER.C6, HEK 293.

In a preferred embodiment, the amino acid residue at positions 40H, 60Hand 61H will be substituted as described supra.

In other embodiments, the amino acid residues at position 40H and 60H or40H and 61H or 60H and 61H will be substituted as described supra.

In still other embodiments, the amino acid residues at position 40H or60H or 61H will be substituted as described supra.

In a preferred embodiment, the method of the invention will result in anantibody with increased expression levels and/or purification yieldsfrom a host cell.

In a more preferred embodiment, the method of the invention will resultin an antibody with increased expression levels and/or purificationyields without negatively affecting antigen binding characteristics.

In a more preferred embodiment, the method of the invention will resultin an antibody with both increased expression levels and/or purificationyields and improved antigen binding characteristics.

The present invention also provides new antibody polypeptides havingmodifications of the heavy chain resulting in improved producibility ascompared to the unmodified antibody.

In a preferred embodiment, the antibodies of the invention have improvedproducibility and little or no reduction in antigen binding. Morepreferably, the antibodies of the invention have both improvedproducibility and improved antigen binding characteristics.

In another embodiment, the heavy chain modifications of the antibodiesof the invention are to residues 40H, 60H, and 61H. Specifically,positions 40H, 60H, and 61H are substituted, where necessary, byalanine, alanine and aspartic acid, respectively.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is the amino acid sequence of the variable regions of the lightchains (VL) (SEQ ID NOS. 1-8) (A) and the heavy (V_(H)) (SEQ ID NOS.9-16 and 24-32) (B) of various antibodies of the invention. Shaded:Positions 40H, 60H and 61H (Kabat numbering); Boxed: CDRs (Kabatdefinition); Each sequence is identified by its name followed by “/M”when the A40/A60/D61 amino acid combination is present in thecorresponding heavy chain. Note: for EA5/M′, only positions A60/D61 arepresent.

FIG. 2 is the binding specificity of the antibodies of the invention asdetermined by surface plasmon resonance detection using a BIAcore 1000instrument. The results for antibodies G5, 1E11, 4C10, 10D3, 12G3 and4B11 and the corresponding substituted antibodies are shown in panel Awhile the results for EA5, MEDI-522 and their corresponding substitutedantibodies are shown in panels B and C respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the substitution ofcertain amino acid residues of the heavy chain of an antibody results ina dramatic increase in the producibility of said antibody in aeukaryotic host cell. In addition, the inventors have also foundunexpectedly, that the amino acid substitutions of the invention did notnegatively affect antigen binding and could actually enhance the antigenbinding properties of the modified antibody. Thus, the inventionincludes antibodies displaying increased producibility wherein bindingaffinity is decreased although still at useful levels, unchanged, orincreased.

Accordingly, the present invention relates to antibodies or antibodyfragments with improved producibility and a method for improving theproducibility of an antibody or antibody fragment by modifying the heavychain. The antibodies or antibody fragments generated by the method ofthe invention will have antigen binding characteristics that are eitherimproved, unchanged, or altered to an acceptable degree. Using methodsdescribed and contemplated herein, the present invention also providesantibodies or antibody fragment comprising said modified heavy chainhaving improved producibility and improved or unchanged antigen bindingcharacteristics.

Without wishing to be bound by any particular theory, the amino acidsubstitutions of the invention improve the producibility of an antibodyor antibody fragment by altering one or more of the factors which limitantibody production in cells including but not limited to, the level ofgene expression, the stability of the messenger RNA, the stability ofthe translated antibody protein, protein folding, level of proteinaggregation, and the toxicity of the antibody to the host cell.

It will be understood that the antibody residue numbers referred toherein are those of Kabat et. al. supra. In addition, the identity ofcertain individual residues at any given Kabat site number may vary fromantibody chain to antibody chain due to interspecies or allelicdivergence. However, for the sake of clarity and simplicity the residuenumbers and identities of the Kabat human IgG heavy chain sequences willbe used herein. Note that complementarity determining regions (CDRs)vary considerably from antibody to antibody (and by definition will notexhibit homology with the Kabat consensus sequences). Maximal alignmentof framework residues frequently requires the insertion of “spacer”residues in the numbering system, to be used for the Fv region. It willbe understood that the CDRs referred to herein are those of Kabat et al.supra.

In the case where there are two or more definitions of a term that areused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term “CDR” todescribe the non-contiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Thisparticular region has been described by Kabat et al., 1991, NIHPublication 91-3242, National Technical Information Service,Springfield, Va.A) and by Chothia et al. (1987, J. Mol. Biol.196:901-17) and additionally by MacCallum et al. (1996, J. Mol. Biol.262:732-45), each of which are incorporated herein by reference, wherethe definitions include overlapping or subsets of amino acid residueswhen compared against each other. Nevertheless, application of eitherdefinition to refer to a CDR of an antibody or variants thereof isintended to be within the scope of the term as defined and used herein.The appropriate amino acid residues that encompass the CDRs as definedby each of the above cited references are set forth below in Table 1 asa comparison. The exact residue numbers which encompass a particular CDRwill vary depending on the sequence and size of the CDR.

Those skilled in the art can routinely determine which residues comprisea particular CDR given the variable region amino acid sequence of theantibody.

TABLE 1 CDR Definitions Kabat¹ Chothia² MacCallum³ VH CDR1 31-35 26-3230-35 VH CDR2 50-65 53-55 47-58 VH CDR3  95-102  96-101  93-101 VL CDR124-34 26-32 30-36 VL CDR2 50-56 50-52 46-55 VL, CDR3 89-97 91-96 89-96¹Residue numbering follows the nomenclature of Kabat et al., supra²Residue numbering follows the nomenclature of Chothia et al., supra³Residue numbering follows the nomenclature of MacCallum et al., supra

In one embodiment, antibodies of the invention will have at least oneamino acid substitution wherein said substituted antibody has increasedproduction levels compared to the antibody without said substitution.

In a specific embodiment, antibodies of the invention are substituted atone or more positions from the group consisting of: 40H, 60H, and 61H,utilizing the numbering system set forth in Kabat. More specifically,one or more of the amino acid residues 40H, 60H and 61H are substitutedwith alanine, alanine and aspartic acid, respectively.

In another embodiment, the invention provides a method for producing asubstituted antibody with increased production levels.

In a preferred embodiment, the invention provides a method forincreasing the producibility of an antibody or antibody fragmentcomprising the steps of: (a) substituting where necessary the amino acidresidues at positions 40H, 60H, and 61H, utilizing the numbering systemset forth in Kabat, of the antibody of interest with alanine, alanineand aspartic acid, respectively; and (b) cultivating the host cell underconditions where the modified antibody polypeptide is expressed by saidhost cell.

It is specifically contemplated that one may choose to analyze thenature of the amino acids at positions 40H, 60H, and 61H prior to makingany substitutions.

One skilled in the art would appreciate that in some cases the antibodyof interest will already have the appropriate sequence at one or more ofthe aforementioned positions. In this situation, substitution(s) willonly be introduced at the remaining non matching position(s) (e.g., atpositions 40H/60H, 40H/61H, 60H/61H, 40H, 60H, or 61H).

In a preferred embodiment, the amino acid residue at positions 40H, 60Hand 61H will be substituted with alanine, alanine and aspartic acidrespectively.

In other preferred embodiments, the amino acid residues at position 40Hand 60H will be substituted with alanine or 40H and 61H will besubstituted with alanine and aspartic acid respectively or 60H and 61Hwill be substituted with alanine and aspartic acid respectively.

In still other preferred embodiments, the amino acid residues atposition 40H or 60H will be substituted with alanine or 61H will besubstituted with aspartic acid.

It is specifically contemplated that conservative amino acidsubstitutions may be made for said amino acid substitutions at positions40H, 60H and/or 61H of the antibody of interest, described supra. It iswell known in the art that “conservative amino acid substitution” refersto amino acid substitutions that substitute functionally-equivalentamino acids. Conservative amino acid changes result in silent changes inthe amino acid sequence of the resulting peptide. For example, one ormore amino acids of a similar polarity act as functional equivalents andresult in a silent alteration within the amino acid sequence of thepeptide. Substitutions that are charge neutral and which replace aresidue with a smaller residue may also be considered “conservativesubstitutions” even if the residues are in different groups (e.g.,replacement of phenylalanine with the smaller isoleucine). Families ofamino acid residues having similar side chains have been defined in theart. Several families of conservative amino acid substitutions are shownin Table 2.

TABLE 2 Families of Conservative Amino Acid Substitutions Family AminoAcids non-polar Trp, Phe, Met, Leu, Ile, Val, Ala, Pro uncharged polarGly, Ser, Thr, Asn, Gln, Tyr, Cys acidic/negatively charged Asp, Glubasic/positively charged Arg, Lys, His Beta-branched Thr, Val, Ileresidues that influence Gly, Pro chain orientation aromatic Trp, Tyr,Phe, His

The term “conservative amino acid substitution” also refers to the useof amino acid analogs or variants. Guidance concerning how to makephenotypically silent amino acid substitutions is provided in Bowie etal. , “Deciphering the Message in Protein Sequences: Tolerance to AminoAcid Substitutions,” (1990, Science 247:1306-10).

In still another preferred embodiment, the method of the invention willresult in an antibody with increased expression levels and/orpurification yields.

In a more preferred embodiment, the method of the invention will resultin an increase in antibody expression levels in crude media samples asdetermined by ELISA and/or purified antibody yields of at least 2 fold,or of at least 4 fold, or of at least 5 fold, or of at least 10 fold, orof at least 25 fold, or of at least 50 fold or of at least 100 fold whencompared to the antibody without said substitution.

One skilled in the art will understand that amino acid substitutions andother modifications of an antibody may alter its antigen bindingcharacteristics (examples of binding characteristics include but are notlimited to, binding specificity, equilibrium dissociation constant(K_(D)), dissociation and association rates (K_(off) and K_(on)respectively), binding affinity and/or avidity) and that certainalterations are more or less desirable. For example a modification thatpreserves or enhances antigen binding would be more preferable then onethat diminished or altered antigen binding. The binding characteristicsof an antibody for a target antigen, may be determined by a variety ofmethods including but not limited it, equilibrium methods (e.g.,enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)),or kinetics (e.g., BIACORE® analysis; see Example 2), for example. Othercommonly used methods to examine the binding characteristics ofantibodies are described in Using Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY, Harrow et al., 1999 and Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow etal., 1989.

It is well known in the art that the equilibrium dissociation constant(K_(D)) is defined as k_(off)/k_(on). It is generally understood that anantibody with a low K_(D) is preferable to an antibody with a highK_(D). However, in some instances the value of the k_(on) or k_(off) maybe more relevant than the value of the K_(D). One skilled in the art candetermine which kinetic parameter is most important for a given antibodyand application. In a preferred embodiment, the method of the inventionwill result in antibodies with improved producibility and one or moreantigen binding characteristics (e.g., binding specificity, K_(D),K_(off), K_(on), binding affinity and/or avidity) that are improved byat least 2%, or by at least 5%, or by at least 10%, or by at least 20%,or by at least 30%, or by at least 40%, or by at least 50%, or by atleast 60%, or by at least 70%, or by at least 80% when compared tokinetic parameters of the antibody without said substitution.

In another embodiment, the method of the invention will result inantibodies with at least one amino acid residue substitution thatincrease expression levels and/or purification yields, but do notsubstantially diminish the antigen binding of the antibody. For example,the method of the invention will generate antibodies that exhibitincrease expression levels and/or purification yields, but preferablyhave no reduction in any antigen binding characteristic (e.g., bindingspecificity, K_(D), K_(off), K_(on), binding affinity and/or avidity),or have one or more antigen binding characteristics that are reduced byless than 1%, or by less than 5%, or by less than 10%, or by less than20%, or by less than 30%, or by less than 40%, or by less than 50%, orby less than 60%, or by less than 70%, or by less than 80% when comparedto antigen binding of the antibody without said substitution.

The skilled artisan will further appreciate that the method of theinvention may also be combined with other methods to increase theproducibility of an antibody. Such methods include but are not limitedto, manipulation of the growth media and/or conditions, modifications ofthe host cell, the introduction of additional amino acid substitutionsor mutations into the heavy and/or light chains of the antibody andother modifications of the antibody. Additionally, the method of theinvention may be combined with additional methods to generate anantibody with other preferred characteristics including but not limitedto: increased serum half life, increase binding affinity, reducedimmunogenicity, increased production, and altered binding specificity(for examples see infra).

The present invention also provides new antibody polypeptides having atleast one amino acid residue substitution that results in improvedproducibility in host cells as compared to the antibody without saidsubstitution.

The present invention further provides new antibody polypeptides havingat least one amino acid residue substitution that results in improvedproducibility in host cells and improvements in one or more antigenbinding characteristics (e.g., binding specificity, K_(D), K_(off),K_(on), binding affinity and/or avidity) as compared to the antibodywithout said substitution.

In a preferred embodiment, the invention refers to antibody polypeptideshaving at least one amino acid residue substitution, characterized inthat their expression levels in crude media samples as determined byELISA and/or purified antibody yields exceed the expression levelsand/or purification yields of the chimeric antibodies withoutsubstitutions by at least 100 fold, or by at least 50 fold, or by atleast 25 fold, or by least 10 fold, or by at least 5 fold, or by atleast 4 fold, or by at least 2 fold.

In a preferred embodiment, antibodies of the invention have bothimproved producibility and one or more antigen binding characteristics(e.g., binding specificity, K_(D), K_(off), K_(on), binding affinityand/or avidity) that are improved by at least 2%, or by at least 5%, orby at least 10%, or by at least 20%, or by at least 30%, or by at least40%, or by at least 50%, or by at least 60%, or by at least 70%, or byat least 80% when compared to kinetic parameters of the antibody withoutsaid substitutions.

In another embodiment, antibodies of the invention will exhibitincreased expression levels and/or purification yields, but preferablyhave no reduction in any antigen binding characteristic (e.g., bindingspecificity, K_(D), K_(off), K_(on), binding affinity and/or avidity),or have one or more antigen binding characteristics that are reduced byless than 1%, or by less than 5%, or by less than 10%, or by less than20%, or by less than 30%, or by less than 40%, or by less than 50%, orby less than 60%, or by less than 70%, or by less than 80% when comparedto antigen binding of the antibody without said substitution.

It is also specifically contemplated that the modified antibodies of theinvention may contain inter alia additional amino acid residuesubstitutions, mutations and/or modifications which result in anantibody with preferred characteristics including but not limited to:increased serum half life, increase binding affinity, reducedimmunogenicity, increased production, and binding specificity (forexamples see infra).

In one embodiment, the modified antibodies of the invention may beengineered to include modifications within the Fc region, typically toalter one or more functional properties of the antibody, such as serumhalf-life, complement fixation, Fc receptor binding, and/orantigen-dependent cellular cytotoxicity. Furthermore, an antibody of theinvention may be chemically modified (e.g., one or more chemicalmoieties can be attached to the antibody) or be modified to alter it'sglycosylation, again to alter one or more functional properties of theantibody. Each of these embodiments is described in further detailbelow. The numbering of residues in the Fc region is that of the EUindex of Kabat.

In one embodiment, the amino acid sequence of the Fc region is modifiedby deleting, adding and/or substituting at least amino acid residue toalter one or more of the functional properties of the antibody describedabove. This approach is described further in Duncan et al, 1988, Nature332:563-564; Lund et al., 1991, J. Immunol 147:2657-2662; Lund et al,1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA92:11980-11984; Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund etal., 1995, Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett54:101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al.,1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol164:4178-4184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et al.,2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,194,551; 6,737,056 U.S. patent application Ser. No. 10/370,749 and PCTPublications WO 94/2935; WO 99/58572; WO 00/42072; WO 04/029207, each ofwhich is incorporated herein by reference in its entirety.

In still another embodiment, the glycosylation of the modifiedantibodies of the invention is modified. For example, an aglycoslatedantibody can be made (i.e., the antibody lacks glycosylation).Glycosylation can be altered to, for example, increase the affinity ofthe antibody for antigen. Such carbohydrate modifications can beaccomplished by, for example, altering one or more sites ofglycosylation within the antibody sequence. For example, one or moreamino acid substitutions can be made that result in elimination of oneor more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. Such an approach is describedin further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861, each ofwhich is incorporated herein by reference in its entirety.

Additionally or alternatively, a modified antibody of the invention canbe made that has an altered type of glycosylation, such as ahypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of whichis incorporated herein by reference in its entirety.

Preferred Antibodies of the Invention

Antibodies modified by the method of the present invention and generatedby the method of the invention may include, but are not limited to,synthetic antibodies, monoclonal antibodies, recombinantly producedantibodies, intrabodies, multispecific antibodies, bispecificantibodies, human antibodies, humanized antibodies, chimeric antibodies,synthetic antibodies, single-chain Fvs (scFv), Fab fragments, F(ab′)fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Inparticular, antibodies used in the methods of the present inventioninclude immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules. The immunoglobulin molecules of the inventioncan be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass of immunoglobulinmolecule.

Antibodies or antibody fragments modified by the method of the inventionand generated by the method of the present invention may be from anyanimal origin including birds and mammals (e.g., human, murine, donkey,sheep, rabbit, goat, guinea pig, camel, horse, or chicken). Preferably,the antibodies are human or humanized monoclonal antibodies. As usedherein, “human” antibodies include antibodies having the amino acidsequence of a human immunoglobulin and include antibodies isolated fromhuman immunoglobulin libraries or from mice that express antibodies fromhuman genes.

Antibodies or antibody fragments modified by the method of the inventionand generated by the method of the present invention may bemonospecific, bispecific, trispecific or of greater multispecificity.Multispecific antibodies may immunospecifically bind to differentepitopes of desired target molecule or may immunospecifically bind toboth the target molecule as well as a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g.,International Publication Nos. WO 93/17715, WO 92/08802, WO 91/00360,and WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Pat.Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; andKostelny et al., 1992, J. Immunol. 148:1547-1553.

The method and antibodies of the present invention encompasses singledomain antibodies, including camelized single domain antibodies (seee.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall etal., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999,J. Immunol. Meth. 231:25; International Publication Nos. WO 94/04678 andWO 94/25591; U.S. Pat. No. 6,005,079; which are incorporated herein byreference in their entireties).

The method and antibodies of the present invention also encompass theuse of antibodies or fragments thereof that have half-lives (e.g., serumhalf-lives) in a mammal, preferably a human, of greater than 15 days,preferably greater than 20 days, greater than 25 days, greater than 30days, greater than 35 days, greater than 40 days, greater than 45 days,greater than 2 months, greater than 3 months, greater than 4 months, orgreater than 5 months. The increased half-lives of the antibodies of thepresent invention or fragments thereof in a mammal, preferably a human,results in a higher serum titer of said antibodies or antibody fragmentsin the mammal, and thus, reduces the frequency of the administration ofsaid antibodies or antibody fragments and/or reduces the concentrationof said antibodies or antibody fragments to be administered. Antibodiesor fragments thereof having increased in vivo half-lives can begenerated by techniques known to those of skill in the art. For example,antibodies or fragments thereof with increased in vivo half-lives can begenerated by modifying (e.g., substituting, deleting or adding) aminoacid residues identified as involved in the interaction between the Fcdomain and the FcRn receptor (see, e.g., International Publication No.WO 97/34631 and U.S. patent application Ser. No. 10/020,354, both ofwhich are incorporated herein by reference in their entireties).

The method and antibodies of the present invention also encompassesantibodies that are bispecific comprising a modified antibody of theinvention, or antigen-binding portion thereof, linked to a secondfunctional moiety having a different binding specificity than saidantibody, or antigen binding portion thereof, of the invention. In afurther embodiment, the invention encompasses antibodies which aremultispecific, where the antibody molecule further comprises a third, ora fourth, or more function moiety having a different binding specificitythan said antibody of the invention, or antigen binding portion thereof.

In a specific embodiment, method and antibodies of the present inventionare bispecific T cell engagers (BiTEs). Bispecific T cell engagers(BiTE) are bispecific antibodies that can redirect T cells forantigen-specific elimination of targets. A BiTE molecule has anantigen-binding domain that binds to a T cell antigen (e.g. CD3) at oneend of the molecule and an antigen binding domain that will bind to anantigen on the target cell. A BiTE molecule was recently described in WO99/54440, which is herein incorporated by reference. This publicationdescribes a novel single-chain multifunctional polypeptide thatcomprises binding sites for the CD19 and CD3 antigens (CD19×CD3). Thismolecule was derived from two antibodies, one that binds to CD19 on theB cell and an antibody that binds to CD3 on the T cells. The variableregions of these different antibodies are linked by a polypeptidesequence, thus creating a single molecule. Also described, is thelinking of the variable heavy chain (VH) and light chain (VL) of aspecific binding domain with a flexible linker to create a single chain,bispecific antibody.

In another embodiment, the BiTE molecule can comprise a molecule thatbinds to other T cell antigens (other than CD3). For example, ligandsand/or antibodies that immunospecifically bind to T-cell antigens likeCD2, CD4, CD8, CD11a, TCR, and CD28 are contemplated to be part of thisinvention. This list is not meant to be exhaustive but only toillustrate that other molecules that can immunospecifically bind to a Tcell antigen can be used as part of a BiTE molecule. These molecules caninclude the VH and/or VL portions of the antibody or natural ligands(for example LFA3 whose natural ligand is CD3).

Methods of Generating Antibodies

Antibodies or antibody fragments modified by the method of the inventionand generated by the invention can be generated by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Monoclonal antibodies modified by the method of the present inventioncan be prepared using a wide variety of techniques known in the artincluding the use of hybridoma, recombinant, and phage displaytechnologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Antibodies: A LaboratoryManual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory Press(Cold Spring Harbor, N.Y., 1988); and Hammerling, et al., in: MonoclonalAntibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (saidreferences incorporated by reference in their entireties). The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. Briefly,mice can be immunized with a antigen of interest, generally but notalways a polypeptide such as a full length protein or a domain thereof(e.g., the extracellular domain) can be utilized, and once an immuneresponse is detected, e.g., antibodies specific for the antigen ofinterest are detected in the mouse serum, the mouse spleen is harvestedand splenocytes isolated. The splenocytes are then fused by well knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. Additionally, a RIMMS (repetitive immunization,multiple sites) technique can be used to immunize an animal (Kilpatricket al., 1997, Hybridoma 16:381-9, incorporated herein by reference inits entirety). Hybridoma clones are then assayed by methods known in theart for cells that secrete antibodies capable of binding a polypeptideof the invention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, monoclonal antibodies can be generated by culturing ahybridoma cell secreting an antibody of interest wherein, preferably,the hybridoma is generated by fusing splenocytes isolated from a mouseimmunized with polypeptide of interest or fragment thereof with myelomacells and then screening the hybridomas resulting from the fusion forhybridoma clones that secrete an antibody able to bind the polypeptideof interest.

Antibody fragments of the invention may be generated by any techniqueknown to those of skill in the art. For example, Fab and F(ab′)2fragments of the invention may be produced by proteolytic cleavage ofimmunoglobulin molecules, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragmentscontain the variable region, the light chain constant region and the CH1domain of the heavy chain. Further, the antibodies of the presentinvention can also be generated using various phage display methodsknown in the art.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles that carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding V_(H) and V_(L)domains are amplified from animal cDNA libraries (e.g., human or murinecDNA libraries of lymphoid tissues). The DNA encoding the V_(H) andV_(L) domains are recombined together with an scFv linker by PCR andcloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). Thevector is electroporated in E. coli and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13 and the V_(H) and V_(L) domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to the antigen epitopeof interest can be selected or identified with antigen, e.g., usinglabeled antigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J.Immunol. Methods 184:177; Kettleborough et al., 1994, Eur. J. Immunol.24:952-958; Persic et al., 1997, Gene 187:9; Burton et al., 1994,Advances in Immunology 57:191-280; International Application No.PCT/GB91/01134; International Publication Nos. WO 90/02809, WO 91/10737,WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, andW097/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484,5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908,5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of whichis incorporated herein by reference in its entirety.

In a preferred embodiment, after phage selection, the antibody codingregions from the phage are isolated and used to generate wholeantibodies, including human antibodies as described in the abovereferences. In another preferred embodiment the reconstituted antibodyof the invention is expressed in any desired host, including bacteria,insect cells, plant cells, yeast, and in particular, mammalian cells(e.g., as described below). Techniques to recombinantly produce Fab,Fab' and F(ab′)2 fragments can also be employed using methods known inthe art such as those disclosed in International Publication No. WO92/22324; Mullinax et al., 1992, BioTechniques 12:864; Sawai et al.,1995, AJRI 34:26; and Better et al., 1988, Science 240:1041 (saidreferences incorporated by reference in their entireties).

The nucleotide sequence encoding an antibody of the invention can beobtained from sequencing hybridoma clone DNA. If a clone containing anucleic acid encoding a particular antibody or an epitope-bindingfragment thereof is not available, but the sequence of the antibodymolecule or epitope-binding fragment thereof is known, a nucleic acidencoding the immunoglobulin may be chemically synthesized or obtainedfrom a suitable source (e.g., an antibody cDNA library, or a cDNAlibrary generated from, or nucleic acid, preferably poly A+ RNA,isolated from any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody) by PCR amplificationusing synthetic primers that hybridize to the 3′ and 5′ ends of thesequence or by cloning using an oligonucleotide probe specific for theparticular gene sequence to identify, e.g., a cDNA clone from a cDNAlibrary that encodes the antibody. Amplified nucleic acids generated byPCR may then be cloned into replicable cloning vectors using any methodwell known in the art.

Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences, e.g.recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see,Or example, the techniques described in Current Protocols in MolecularBiology, F. M. Ausubel et al., ed., John Wiley & Sons (Chichester,England, 1998); Molecular Cloning: A Laboratory Manual, 3nd Edition, J.Sambrook et al., ed., Cold Spring Harbor Laboratory Press (Cold SpringHarbor, NY, 2001); Antibodies: A Laboratory Manual, E. Harlow and D.Lane, ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor,N.Y., 1988); and Using Antibodies: A Laboratory Manual, E. Harlow and D.Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y.,1999) which are incorporated by reference herein in their entireties),to generate antibodies having a different amino acid sequence by, forexample, introducing deletions, and/or insertions into desired regionsof the antibodies.

In a preferred embodiment, antibodies of the invention include aminoacid substitutions into the variable region of the heavy chain such thatpositions 41H, 60H and 61H substituted by alanine, alanine and asparticacid, respectively.

In a more preferred embodiment, the V_(H) and V_(L) nucleotide sequencesare cloned and used to generate whole antibodies. Utilizing cloningtechniques known to those skilled in the art, the PCR primers includingV_(H) or V_(L) nucleotide sequences, a restriction site, and a flankingsequence to protect the restriction site are used to amplify the V_(H)or V_(L) sequences in scFv. The PCR amplified V_(H) domains are clonedinto vectors expressing a V_(H) constant region, e.g., the human gamma 4constant region, and the PCR amplified V_(L) domains are cloned intovectors expressing a V_(L) constant region, e.g., human kappa or lambdaconstant regions. The V_(H) and V_(L) domains may also be cloned intoone vector expressing the necessary constant regions. The heavy chainconversion vectors and light chain conversion vectors are thenco-transfected into cell lines to generate stable or transient celllines that express full-length antibodies, e.g., IgG, using techniquesknown to those of skill in the art.

It is specifically contemplated that for some uses, including in vivouse of antibodies in humans and in vitro detection assays, antibodies ofthe invention are preferably human or chimeric antibodies. Completelyhuman antibodies are particularly desirable for therapeutic treatment ofhuman subjects. Human antibodies can be made by a variety of methodsknown in the art including phage display methods described above usingantibody libraries derived from human immunoglobulin sequences. See alsoU.S. Pat. Nos. 4,444,887 and 4,716,111; and International PublicationNos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then be bred to producehomozygous offspring that express human antibodies. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., International Publication Nos. WO 98/24893, WO 96/34096, and WO96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporatedby reference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.) andMedarex (Princeton, N.J.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such as,for example, antibodies having a variable region derived from anon-human antibody and a human immunoglobulin constant region. Methodsfor producing chimeric antibodies are known in the art. See e.g.,Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214;Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Pat.Nos. 5,807,715, 4,816,567, and 4,816,397, CDR-grafting (EP 239,400;International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan, 1991, Molecular Immunology 28(4/5): 489-498; Studnickaet al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, PNAS91:969), and chain shuffling (U.S. Pat. No. 5,565,332). which areincorporated herein by reference in their entirety.

Methods of Expressing Antibodies

Recombinant expression of an antibody requires construction of anexpression vector containing a nucleotide sequence that encodes theantibody. Once a nucleotide sequence encoding an antibody molecule or aheavy or light chain of an antibody, or portion thereof (preferablycontaining the heavy or light chain variable regions) has been obtained,the vector for the production of the antibody molecule may be producedby recombinant DNA technology using techniques well known in the art.Thus, methods for preparing a protein by expression a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods, which are well known to those skilled in the art, canbe used to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.

The invention, thus, provides replicable vectors comprising a nucleotidesequence encoding a modified antibody molecule with one or moremodifications in the amino acid residues 40H, 60H and 61H of the heavychain. The nucleotide sequence encoding the heavy-chain variable region,light-chain variable region, both the heavy-chain and light-chainvariable regions, an epitope-binding fragment of the heavy- and/orlight-chain variable region, or one or more complementarily determiningregions (CDRs) of an antibody may be cloned into such a vector forexpression.

The antibody expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce a substituted antibody have improvedproducibility. A variety of host-expression vector systems may beutilized to express the antibody molecules of the invention. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences express an antibody molecule ofthe invention in situ.

In a preferred embodiment, antibodies generated by the method of theinvention are expressed in eukaryotic host cells. In a more preferredembodiment the host cell is mammalian. Mammalian cell systems includebut are not limited to, CHO, BHK, HeLa, COS, MDCK, NIH 3T3, W138, NSO,SP2/0 and other lymphocytic cells, and human cells such as PER.C6, HEK293 harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). For example, mammalian cells such asChinese hamster ovary cells (CHO) in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene, 45:101; and Cockett et al., 1990,BioTechnology, 8:2).

In mammalian host cells, a number of viral-based expression systems maybe utilized to express an antibody molecule of the invention. In caseswhere an adenovirus is used as an expression vector, the antibody codingsequence of interest may be ligated to an adenovirustranscription/translation control complex, (e.g., the late promoter andtripartite leader sequence). This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe antibody molecule in infected hosts (e.g. see Logan & Shenk, 1984,Proc. Natl. Acad. Sci. USA, 1:355-59). Specific initiation signals mayalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter et al., 1987, Methods in Enzymol.,153:516-44).

In addition, a host cell strain may be chosen which modulates theexpression of the antibody sequences, or modifies and processes theantibody in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the antibody. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the antibody expressed. To this end, itis specifically contemplated that eukaryotic host cells which possessthe cellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product are be used. Suchmammalian host cells include but are not limited to , CHO, BHK, HeLa,COS, MDCK, NIH 3T3, W138, NSO, SP2/0 and other lymphocytic cells, andhuman cells such as PER.C6, HEK 293.

For long-term, high-yield production of recombinant antibodies, stableexpression is preferred. For example, cell lines that stably express theantibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators,—85 polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell,11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA, 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell, 22:8-17) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1908, Proc. Natl. Acad. Sci. USA, 77:357 and O'Hare et al.,1981, Proc. Natl. Acad. Sci. USA, 78:1527), gpt, which confersresistance to mycophenolic acid (Mulligan & Berg, 1981. Proc. Natl. Acad. Sci. USA, 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Wu and Wu, 1991, Biotherapy, 3:87-95; Tolstoshev,1993, Ann. Rev. Pharmacol. Toxicol., 32:573-96; Mulligan, 1993, Science,260:926-32; and Morgan and Anderson, 1993, Ann. Rev. Biochem., 62:191-217; and May, 1993, TIB TECH, 11(5):155-2); and hygro, which confersresistance to hygromycin (Santerre et al., 1984, Gene, 30:147). Methodscommonly known in the art of recombinant DNA technology may be routinelyapplied to select the desired recombinant clone, and such methods aredescribed, for example, in Ausubel, F. M., et al.,1998, CurrentProtocols in Molecular Biology, John Wiley & Sons, and Sambrook, etal.,2001, Molecular Cloning: A Laboratory Manual, 3nd Edition, which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be further increasedby vector amplification (for a review, see Bebbington and Hentschel,1987, The use of vectors based on gene amplification for the expressionof cloned genes in mammalian cells in DNA cloning, Vol. 3. AcademicPress, New York). When a marker in the vector system expressing antibodyis amplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.,3:257)

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers, which enable equalexpression of heavy and light chain polypeptides.

Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature,322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA, 77:2 197). Thecoding sequences for the heavy and light chains may comprise cDNA orgenomic DNA.

In one embodiment, the whole recombinant antibody molecule, isexpressed. In another embodiment, fragments (e.g., Fab fragments, F(ab′)fragments, and epitope-binding fragments) of the immunoglobulin moleculeare expressed.

Once an antibody molecule of the invention has been produced byrecombinant expression, it may be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A purification, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard techniques for the purification of proteins. Further, theantibodies of the present invention or fragments thereof may be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

Antibody Derivatives

Antibodies modified by the method of the present invention and generatedby the method of the invention include derivatives that are modified(i.e., by the covalent attachment of any type of molecule to theantibody such that covalent attachment). For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to, specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

Antibodies or fragments thereof with increased in vivo half-lives can begenerated by attaching to said antibodies or antibody fragments polymermolecules such as high molecular weight polyethylene glycol (PEG). PEGcan be attached to said antibodies or antibody fragments with or withouta multifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of said antibodies or antibody fragments orvia epsilon-amino groups present on lysine residues. Linear or branchedpolymer derivatization that results in minimal loss of biologicalactivity will be used. The degree of conjugation will be closelymonitored by SDS-PAGE and mass spectrometry to ensure proper conjugationof PEG molecules to the antibodies. Unreacted PEG can be separated fromantibody-PEG conjugates by, e.g., size exclusion or ion-exchangechromatography.

The present invention encompasses antibodies modified by the method ofthe present invention and generated by the method of the invention (orfragments thereof) recombinantly fused or chemically conjugated(including both covalent and non-covalent conjugations) to aheterologous polypeptide (or portion thereof, preferably to apolypeptide of at least 10, at least 20, at least 30, at least 40, atleast 50, at least 60, at least 70, at least 80, at least 90 or at least100 amino acids) to generate fusion proteins. The fusion does notnecessarily need to be direct, but may occur through linker sequences.For example, antibodies may be used to target heterologous polypeptidesto particular cell types, either in vitro or in vivo, by fusing orconjugating the antibodies to antibodies specific for particular cellsurface receptors. Antibodies fused or conjugated to heterologouspolypeptides may also be used in in vitro immunoassays and purificationmethods using methods known in the art. See e.g., InternationalPublication WO 93/21232; EP 439,095; Naramura et al., 1994, Immunol.Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452, whichare incorporated by reference in their entireties.

The present invention further includes compositions comprisingheterologous polypeptides fused or conjugated to antibody fragments. Forexample, the heterologous polypeptides may be fused or conjugated to aFab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment, or portionthereof. Methods for fusing or conjugating polypeptides to antibodyportions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603,5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EP 307,434;EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570;Ashkenazi et al., 1991, PNAS 88: 10535-10539; Zheng et al., 1995, J.Immunol. 154:5590-5600; and Vil et al., 1992, PNAS 89:11337-11341 (saidreferences incorporated by reference in their entireties).

DNA shuffling may be employed to alter the activities of antibodies ofthe invention or fragments thereof (e.g., antibodies or fragmentsthereof with higher affinities and lower dissociation rates). See,generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252;and 5,837,458, and Patten et al., 1997, Curr. Opinion Biotechnol.8:724-33; Harayama, 1998, Trends Biotechnol. 16:76; Hansson, et al.,1999, J. Mol. Biol. 287:265; and Lorenzo and Blasco, 1998, BioTechniques24:308 (each of these patents and publications are hereby incorporatedby reference in its entirety). Antibodies or fragments thereof, or theencoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination.

Moreover, the antibodies or fragments thereof can be fused to markersequences, such as a peptide to facilitate purification. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., 1989, PNAS 86:821,for instance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the hemagglutinin “HA” tag, which corresponds to anepitope derived from the influenza hemagglutinin protein (Wilson et al.,1984, Cell 37:767) and the “flag” tag.

In other embodiments, antibodies modified by the method of the presentinvention and generated by the method of the invention or fragments orvariants thereof can be conjugated to a diagnostic or detectable agent.Such antibodies can be useful for monitoring or prognosing thedevelopment or progression of a cancer as part of a clinical testingprocedure, such as determining the efficacy of a particular therapy.Such diagnosis and detection can accomplished by coupling the antibodyto detectable substances including, but not limited to various enzymes,such as but not limited to horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic groups, such asbut not limited to streptavidin/biotin and avidin/biotin; fluorescentmaterials, such as but not limited to, umbelliferone, fluorescein,fluorescein isothiocynate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; luminescent materials,such as but not limited to, luminol; bioluminescent materials, such asbut not limited to, luciferase, luciferin, and aequorin; radioactivematerials, such as but not limited to, bismuth (²¹³Bi), carbon (¹⁴C),chromium (⁵¹Cr), cobalt (⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd,¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga), germanium (⁶⁸Ge), holmium (166Ho), indium(¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I),lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu), manganese (⁵⁴Mn), molybdenum(⁹⁹Mo), palladium (103Pd), phosphorous (³²P), praseodymium (¹⁴²Pr),promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re), rhodium (¹⁰⁵Rh), ruthemium(⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc), selenium (⁷⁵Se), strontium(⁸⁵Sr), sulfur (³⁵S), technetium (⁹⁹Tc), thallium (²⁰¹Ti), tin (¹¹³Sn,¹¹⁷Sn), tritium (³H), xenon (¹³³Xe), ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium(⁹⁰Y), zinc (⁶⁵Zn); positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions.

The present invention further encompasses uses of modified antibodies ofthe invention or fragments thereof conjugated to a therapeutic agent.

An antibody or fragment thereof may be conjugated to a therapeuticmoiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion, e.g., alpha-emitters. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxel, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, epirubicin, and cyclophosphamide and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Further, an antibody or fragment thereof may be conjugated to atherapeutic agent or drug moiety that modifies a given biologicalresponse. Therapeutic agents or drug moieties are not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, Onconase (or another cytoxic RNase), pseudomonas exotoxin,cholera toxin, or diphtheria toxin; a protein such as tumor necrosisfactor, α-interferon, β-interferon, nerve growth factor, plateletderived growth factor, tissue plasminogen activator, an apoptotic agent,e.g., TNF-α, TNF-β, AIM I (see, International Publication No. WO97/33899), AIM II (see, International Publication No. WO 97/34911), FasLigand (Takahashi et al., 1994, J. Immunol., 6:1567), and VEGI (see,International Publication No. WO 99/23105), a thrombotic agent or ananti-angiogenic agent, e.g., angiostatin or endostatin; or, a biologicalresponse modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), or a growth factor(e.g., growth hormone (“GH”)).

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive materials or macrocyclic chelators useful for conjugatingradiometal ions (see above for examples of radioactive materials). Incertain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug.Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Examples

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations which become evident as a result of the teachings providedherein.

Example 1 Generation and Expression of the Various Antibody Constructs

Six humanized monoclonal antibodies (G5, 10D3, 12G3, 1E11, 4C10, 4B11)and one human/mouse chimeric antibody (EA5) were generated against acommon antigen, EphA2. All of these antibodies were poorly expressed inmammalian cells (see Table 3). Another humanized antibody, MEDI-522,which is expressed well in mammalian cells (see Table 3) was also usedin these studies. One or more heavy chain substitutions at positions 40,60 and/or 61 were generated in each of these antibodies to determine theeffect on producibility by the presence of one or more preferred aminoacid residues at these positions. Six of the humanized antibodiescontained an Alanine at position H40, these antibodies were substitutedwith Alanine and Aspartate at positions H60 and H61 respectively. Thelast humanized antibody, MEDI-522 had both the H40 and H61 preferredamino acids, here position H60 was substituted with Alanine. Thechimeric antibody, EA5, against the same antigen did not contain any ofthe preferred amino acids at positions H40, H60 or H61. Two separateheavy chains were generated for EA5, one which contained substitutionsat positions 60 and 61 and another which contained substitutions atpositions H40, H60 and H61. The specific amino acid residues of theheavy chain that were modified (see FIG. 1B) are described below. In allcases substitutions resulting in one or more preferred heavy chainresidues at positions 40, 60 and 61 resulted in improved producibility(see Table 3). Interestingly, in the case of EA5 which contained none ofthe preferred amino acids, the heavy chain A60/D61 combination (EA5/M’SEQ ID NO.: 31) by itself significantly increased production yields.

Materials and Methods

Generation, Characterization and Cloning of Antigen Specific Antibodies:General methods for generating, screening, cloning and expressingantibodies are known to practitioners of the art. See, e.g., CurrentProtocols in Molecular Biology, F. M. Ausubel et al., ed., John Wiley &Sons (Chichester, England, 1998); Molecular Cloning: A LaboratoryManual, 3nd Edition, J. Sambrook et al., ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y., 2001); Antibodies: ALaboratory Manual, E. Harlow and D. Lane, ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y., 1988); and Using Antibodies:A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring HarborLaboratory (Cold Spring Harbor, N.Y., 1999) which are incorporated byreference herein in their entireties.

Generation Of Heavychain Substitutions: The variable regions of thelight chains of antibody clones G5, 10D3, 12G3, 1E11, 4C10, 4B11,MEDI522 and EA5 (SEQ ID NOS. 1-8, respectively) and the variable regionsof the heavy chains of antibody clones G5, 10D3, 12G3, 1E11, 4C10, 4B11,MEDI522 and EA5 (SEQ ID NOS. 9-16, respectively) were individuallycloned into mammalian expression vectors encoding a humancytomegalovirus major immediate early (hCMVie) enhancer, promoter and5′-untranslated region (Boshart et al., 1985, Cell 41:521-30). In thissystem, a human yl chain is secreted along with a human κ chain (Johnsonet al., 1997, J. Infect. Dis. 176:1215-24). All of the heavy chainsubstitutions were introduced by site-directed mutagenesis using a QuickChange Multi Mutagenesis Kit (Stratagene, Calif.) according to themanufacturer's instructions. Specifically, S60A/A61D were introducedinto clones G5, 10D3, 12G3, 1E11, 4C10 and 4B11 using the primer:5′-ACACAACAGAGTACGCTGACTCTGTGAAGGGTAGAG TCACCATT-3′ (SEQ ID NO. 17);this generated heavy chain antibody clones G5/M, 10D3/M, 12G3/M, 1E11/M,4C10/M and 4B11/M (SEQ ID NOS. 24-29); L60A was introduced into MEDI522using the primers: 5′-GGTGGTGGTAGCACCTACTATGCAGACACTGTGCAGGGCCGATTCACC-3′ (SEQ ID NO.: 18) and 5′-GGTGAATCGGCCCTGCACAGTGTCTGCATAGTAGGTGCTACCACCACC-3′ (SEQ ID NO.: 19) generatingMEDI522/M (SEQ ID NO. 30); N60A/Q61D were introduced into EA5 using theprimers: 5′-GTTACAATGGTGTTACTAGCTACGCCGACAAGTTCAAGGGCAAGG CCAC-3′ (SEQID NO.: 20) and 5′-GTGGCCTTGCCCTTGAACTTGTCGGCGTAGCT AGTAACACCATTGTAAC-3′(SEQ ID NO.: 21) generating EA5/M′ (SEQ ID NO.: 31); and S40A/N60A/Q61Dwere introduced into EA5 using the primers: 5′-CTACATGCACTGGGTCAAGCAGGCCCATGGAAAGAGCCTTGAG-3′ (SEQ ID NO.: 22),5′-CTCAAGGCTCTTTCCATGGGCCTGCTTGACCCAGTGCATGTAG-3′ (SEQ ID NO.: 23),5′-GTTACAATGGTGTTACTAGCTACGCCGACAAGTTCAAGGGCAAGGCCAC-3′ (SEQ ID NO.:20)and 5′-GTGGCCTTGCCCTTGAACTTGTCGGCGTAGCTAGT AACACCATTGTAAC-3′ (SEQ IDNO.: 21) generating EA5/M (SEQ ID NO.: 32). Note that the light chainsremain unaltered and are still represented by SEQ ID NOS 1-8) (FIG. 1A).The sequences were verified using an ABI 3100 sequencer. Human embryonickidney (HEK) 293 cells were then transiently transfected with thevarious antibody constructs in 35 mm, 6-wells dishes using Lipofectamineand standard protocols. Supernatants were harvested twice at 72 and 144hours post-transfection (referred to as 1^(st) and 2^(nd) harvest,respectively). The secreted, soluble human IgG1s were then assayed interms of production yields and binding to original antigen (see below).

Measurement Of The Expression Yields: The expression yields of antibodyclones G5, G5/M, 10D3, 10D3/M, 12G3, 12G3/M, 1E11, 1E11/M, 4C10, 4C10/M,4B11 and 4B11/Mut were measured by ELISA. Transfection supernatantscollected twice at three days intervals (see above) were assayed forantibody production using an anti-human IgG ELISA. Briefly, individualwells of a 96-well Biocoat plate (BD Biosciences, San Jose, Calif.)coated with a goat anti-human IgG were incubated with samples(supernatants) or standards (human IgG, 0.5-100 ng/ml), then with ahorseradish peroxydase conjugate of a goat anti-human IgG antibody.Peroxydase activity was detected with 3,3′,5,5′-tetramethylbenzidine andthe reaction was quenched with 0.2 M H₂SO₄. Plates were read at 450 nm.The results are summarized in Table 3.

TABLE 3 Producibility Improvements of Heavy Chain ModifiedAntibodies^(a) Transfection Transfection Transfection TransfectionTransfection Fold #1 #2 #3 #4 #5 increase^(d) Modified H1^(b) H2^(c) H1H2 H1 H2 H1 H2 H1 H2 H1 H2 Antibody μg/ml μg/ml μg/ml μg/ml μg/ml μg/mlG5 0.3-1.2 0.5-1.3 0.6-1.4 G5/M 1.6-3.8 2.5-6.2 4.4-3.8 1E11 0.7-2.01.2-3.4 1E11/M 1.7-3.3 1.3-3.9 1.6-1.3 4C10 2.0-3.0 2.4-3.2 2.1-3.34C10/M 3.2-5.8 3.8-7.3 5.0-4.6 6.8-7.8 5.1-7.7 2.2-2.1 10D3 0.7-1.71.4-3.5 10D3/M 1.2-2.9 2.8-5.1 2.0-1.5 12G3 0.9-2.3 1.8-3.6 1.4-2.412G3/M N.D. 3.5-8.7 3.2-5.4 3.3-5.9 4.4-8.4 2.6-2.6 4B11 0.4-1.5 0.7-3.04B11/M 1.0-2.3 2.4-5.2 3.0-1.7 MEDI522 14.8-12.2 10.9-8.8  16.9-11.8MEDI522/M 19.3-19.4 18.6-12.3 23.7-16.2 1.4-1.4 EA5 2.7-2.8 1.0-1.24.0-2.9 EA5/M′ 3.3-3.9 1.1-1.9 3.6-5.5 1.1-1.6 EA5/M 4.6-2.4 2.4-2.24.8-3.9 1.5-1.2 ^(a)HEK 293 cells were transiently transfected with thevarious antibody constructs. ^(b)H1 = First Harvest (72 hourspost-transfection). ^(c)H2 = Second Harvest (144 hourspost-transfection). ^(d)Fold increase = average yield for each harvest(H1, H2) of the heavy chain modified “Mut” antibody divided by theaverage yield for each harvest of the unmodified antibody.

Example 2

Analysis of the Binding Characteristics of the Modified Antibodies

Because two of the heavy chain substitutions (positions 60H and 61H,Kabat numbering) fall within the CDRs as defined by Kabat, it waspossible that the general binding characteristics of the substitutedantibodies had been altered. Remarkably, the modifications improved theyields for each of the six antibodies without significantly altering thebinding specificity (see FIGS. 2A-C). Two of the modified antibodieswere chosen for more extensive analysis. Surprisingly, the bindingconstants of one were improved by at least 20%, while the other remainedvirtually unchanged (see Table 4).

Materials and Methods

Binding Specificity via BIAcore Analysis: The interaction of immobilizedEphA2-Fc or αvβ3 with IgG-containing transfection supernatantscorresponding to clones G5, G5/M, 10D3, 10D3/M, 12G3, 12G3/M, 1E11,1E11/M, 4C10, 4C10/M, 4B11 and 4B11/M (FIG. 2A), EA5/EA5M′ (FIG. 2B) andMEDI522/MEDI522M (FIG. 2C) in addition an irrelevant antibody wasincluded. The antibodies were monitored by surface plasmon resonancedetection using a BIAcore 3000 instrument (Pharmacia Biosensor, Uppsala,Sweden). EphA2-Fc and αvβ3 were coupled to the dextran matrix of a CM5sensor chip (Pharmacia Biosensor) using an Amine Coupling Kit asdescribed (Johnsson et al., 1991, Anal. Biochem. 198:268-77) at asurface density of 4539 RU and 4995 RU for EphA2-Fc in FIGS. 2A and 2Brespectively. αvβ3 was couple at a surface density of 4497 RU (FIG. 2C).250 μl of each transfection supernatant (2^(nd) transfection, 2^(nd)harvest for those in FIG. 2A, 2^(nd) transfection, 1^(st) harvest forthose in FIGS. 2B-C) was injected over there respective surfaces. Allbinding experiments were performed at 25° C. at a flow rate of 75μL/min; data were collected for approximately 20 min and one 1-min pulseof 1M NaCl, 50 mM NaOH was used to regenerate the surfaces. The bindingdata for all the antibodies is shown in FIGS. 2A-2C.

Kinetic Analysis via BIAcore: The interaction of soluble 12G3, 4C10,12G3/Mut and 4C10/Mut with immobilized EphA2-Fc was monitored by surfaceplasmon resonance detection using a BIAcore 3000 instrument (PharmaciaBiosensor, Uppsala, Sweden). EphA2-Fc was coupled to the dextran matrixof a CM5 sensor chip (Pharmacia Biosensor) using an Amine Coupling Kitas described (Johnsson et al. supra) at a surface density of 162 RU.IgGs were diluted in 0.01 M HEPES pH 7.4 containing 0.15 M NaCl, 3 mMEDTA and 0.005% P20. All subsequent dilutions were made in the samebuffer. All binding experiments were performed at 25° C. with IgGconcentrations typically ranging from 100 nM to 0.2 nM at a flow rate of75 μL/min; data were collected for approximately 20 min and one 1-minpulse of 1M NaCl, 50 mM NaOH was used to regenerate the surfaces. IgGswere also flowed over an uncoated cell and the sensorgrams from theseblank runs subtracted from those obtained with EphA2-Fc-coupled chips.Data were fitted to a 1:1 Langmuir binding model. This algorithmcalculates both the k_(on), and the k_(off), from which the apparentequilibrium dissociation constant, K_(D), is deduced as the ratio of thetwo rate constants (k_(off)/k_(on)). The data are presented in Table 4.

TABLE 4 Binding Affinities Modified k_(on) k_(off) K_(D) Antibody M⁻¹ ·s⁻¹ s⁻¹ (pM) 4C10 1.02 × 10⁵ 9.75 × 10⁻⁶ 95 4C10/M 6.41 × 10⁴ 5.96 ×10⁻⁶ 93 12G3 2.46 × 10⁵ 8.49 × 10⁻⁶ 34 12G3/M 1.87 × 10⁵ <5.0 × 10^(−6a)<27^(a ) ^(a)Below the limit of detection

Discussion

Despite advances in recombinant antibody engineering and production,expression levels of a given antibody are often disappointing. Areproducible method to increase the producibility of any antibody bydirectly modifying the amino acid sequence of antibody heavy chain wouldbe of significant benefit for the production of numerous therapeuticantibodies.

We have demonstrated for the first time that the specific substitutionof one-three heavy chain residues results in a dramatic increase in theproducibility of the antibody leading to improved production yields.Surprisingly, these same three substitutions reproducibly improved theproducibility of seven different antibodies indicating that the identityof these three heavy chain residues is important for the producibilityof an antibody. In addition we show that the substitution of these heavychain residues does not adversely alter the antigen binding of themodified antibody and can even result in improvements of the antigenbinding characteristics. Furthermore, we show that the presence ofcertain preferred amino acid residues at positions H40, H60 and H61increases the producibility of antibodies containing variable domainsfrom multiple origins including humanized and human-mouse chimericantibodies. Taken together, these results demonstrate that the specificsubstitution, or specific engineering of one or more heavy chainresidues at positions 40, 60 and 61 to improve the producibility of anantibody is widely applicable and can be utilized to increase the yieldsof virtually any antibody.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference in theirentirety for all purposes.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of increasing the production of an antibody from aeukaryotic host cell, wherein said method comprises the steps of: (a)introducing one or more mutations into a nucleotide sequence encodingthe antibody heavy chain, wherein said one or more mutations result inthe substitution of one or more of the amino acid residues selected fromthe group consisting of: positions 40H, 60H, and 61H, utilizing thenumbering system set forth in Kabat, with alanine, alanine, and asparticacid, respectively; (b) introducing into the eukaryotic host cell thenucleotide sequence encoding the heavy chain of the antibody and anucleotide sequence encoding the antibody light chain; and (c)cultivating the eukaryotic host cell under conditions wherein theantibody comprising the substitution is expressed by said eukaryotichost cell, wherein said antibody is a full length antibody; and whereinproduction of the antibody comprising the substitution is increased byat least 1.3 fold and the binding specificity is unchanged compared tothe antibody without the substitution.
 2. The method of claim 1, whereinposition 40H is substituted with alanine
 3. The method of claim 1,wherein position 60H is substituted with alanine
 4. The method of claim1, wherein position 61H is substituted with aspartic acid.
 5. The methodof claim 1, wherein positions 40H and 60H are each substituted withalanine.
 6. The method of claim 1, wherein position 40H and 61H aresubstituted with alanine and aspartic acid, respectively.
 7. The methodof claim 1, wherein position 60H and 61H are substituted with alanineand aspartic acid, respectively.
 8. The method of claim 1, whereinposition 40H, 60H and 61H are substituted with alanine, alanine andaspartic acid, respectively.
 9. The method of claim 1, whereinproduction of the antibody comprising the substitution is increased byat least 1.3 to 15 fold compared to the antibody without thesubstitution.
 10. The method of claim 1, wherein the equilibriumdissociation constant (K_(D)) of the antibody comprising thesubstitution is improved by at least 1%-25% compared to the antibodywithout the substitution.
 11. The method of claim 1, wherein there is anincrease in the equilibrium dissociation constant (K_(D)) of theantibody comprising the substitution of less than 5% compared to theantibody without the substitution.
 12. The method of claim 1, whereinthere is an increase in the equilibrium dissociation constant (K_(D)) ofthe antibody comprising the substitution of less than 5%-60% compared tothe antibody without the substitution
 13. The method of claim 1, whereinthere is no change in the equilibrium dissociation constant (K_(D)) ofthe antibody comprising the substitution compared to the antibodywithout the substitution.
 14. The method of claim 1, wherein there is areduction in the K_(on) rate of the antibody comprising the substitutionof less than 5% compared to the antibody without the substitution. 15.The method of claim 1, wherein there is a reduction in the K_(on) rateof the antibody comprising the substitution of less than 5%-60% comparedto the antibody without the substitution.
 16. The method of claim 1,wherein said eukaryotic host cell is a mammalian cell.
 17. The method ofclaim 14, wherein said mammalian cell is selected from the groupconsisting of: (a) HEK293 cell, (b) NS0 cell, (c) CHO cell, (d) COScell, (e) SP2/0 cell, and (f) PER.C6 cell.
 18. An antibody comprising asubstitution of one or more amino acid residues selected from the groupconsisting of: positions 40H, 60H, and 61H, utilizing the numberingsystem set forth in Kabat, with alanine, alanine, and aspartic acid,respectively; wherein said antibody is a full length antibody; andwherein production of the antibody comprising the substitution in aeukaryotic host cell is increased by at least 1.3 fold and the bindingspecificity is unchanged compared to the antibody without thesubstitution.
 19. The antibody of claim 18, (a) wherein the amino acidresidue a position 40H or 60H is substituted with alanine, or the aminoacid at position 61H is replaced with aspartic acid, or (b) wherein theamino acid residue at positions 40H and 60H are both substituted withalanine, or (c) wherein the amino acid residues at position 40H issubstituted with alanine, and the amino acid residue at position 61H issubstituted with aspartic acid, or (d) wherein the amino acid residuesat position 60H is substituted with alanine, and the amino acid residueat position 61H is substituted with aspartic acid, or (e) wherein theamino acid residues at positions 40H and 60H are substituted withalanine, and the amino acid residue at position 61 H is substituted withaspartic acid.
 20. The antibody of claim 1, wherein the productionlevels are increased by at least 1.3-15 fold.